Efficient delivery of foreign genetic material to fibroblast-like or macrophage-like cells, especially synoviocytes, has proven to be a difficult goal to achieve. Even with the currently developed viral vectors that are, in general, very effective in delivering foreign genetic material to cells, fibroblast-like or macrophage-like cells have been difficult to provide with foreign genetic material. The relative inefficient transduction of these cells, especially of synoviocytes, has hampered the development of therapeutic approaches based on nucleic acid transfer to these cells.
As a result of the inefficient delivery of nucleic acid into, for example, synoviocytes, therapeutic approaches based on nucleic acid transfer involving these cells have focused on strategies in which a low transduction efficiency could at least in part be tolerated. For instance, by relying on delivering nucleic acid encoding proteins with so-called bystander effect, i.e., the expression in a transduced cell of which affects the function of un-transduced cells in the vicinity of transduced cells. Non-limiting examples of proteins with bystander effect are, for instance, excreted factors and/or suicide gene expression such as herpes simplex virus (“HSV”) thymidine kinase (“TK”) expression which in the presence of gancyclovir leads to production of toxic metabolites. The HSV TK-gene encodes a protein capable of metabolizing the relatively non-toxic anti-viral drug gancyclovir (“GCV”) into a mono-phosphorylated product. Subsequent phosphorylation by mammalian kinases results in a tri-phosphorylated nucleoside analogue (“GCV-PPP”) that inhibits DNA-polymerase and kills cells, probably through apoptosis (Vincent et al., 1996).
Although the use of a bystander effect may, in part, reduce the requirement for efficient transduction of fibroblast-like or macrophage-like cells, a more efficient method of transferring genetic material nevertheless is still desirable for economic and safety reasons. Safety aspects include, for instance, the relative sensitivity of liver cells towards toxicity of HSV-TK based cell kill. When cells, other than liver cells, form the target population for suicide by HSV-TK, liver cell transduction should be prevented as much as possible. Unintended liver cell transduction can occur, for instance, through leakage of a nucleic acid delivery vehicle from the site of delivery into the blood stream from where it is transported to the liver. This leakage is dependent on, among other things, the amount of nucleic acid delivery vehicle used. Thus, when, for instance, synoviocytes form the target cells, a certain amount of nucleic acid delivery vehicle will be needed for obtaining a desired level of transduction. When less nucleic acid delivery vehicle is used, leakage of nucleic acid delivery vehicle is less of a problem.
Non-limiting examples in which nucleic acid transfer to fibroblast-like or macrophage-like cells would be beneficial are chronic erosive joint diseases like rheumatoid arthritis, ankylosing spondylitis, and juvenile chronic arthritis. A favorable target cell for nucleic acid transfer in these diseases is the synoviocyte. However, with current methods, the efficiency of transduction of such cells leaves much to be desired.
In a diarthrodial movable joint, smooth articulation is ensured by the unique macromolecular structure of articular cartilage, which covers the end of the bones. The articular cartilages move against one another within a cavity, the joint space, which is lined by a tissue called the synovium. The synovium consists of macrophage-like type A cells and fibroblast-like type B cells, and is underlain by a sparsely cellular subsynovium which, depending on anatomical localization, may be fibrous, adipose or areolar in nature. The fibroblast-like synoviocytes (“FLS”) are distinguishable from normal fibroblast cells in the subintimal synovium by differential gene expression patterns. FLS have been shown to express high levels of uridine diphosphoglucose dehydrogenase (“UDPGD”), high levels of vascular cell adhesion molecule-1 (“VCAM-1”), intercellular adhesion molecule-1 (“ICAM-1”) as well as CD44 (hyaluronic acid receptor), fibronectin receptor and β-integrins. Sublining fibroblasts or fibroblasts from other sources do not, or at a lower level, express these markers (reviewed by J. C. W. Edwards, 1995; G. S. Firestein, 1996).
Rheumatoid arthritis is characterized by massive hyperplasia of the synovium and the presence of inflammatory cells (lymphocytes, macrophages and mast cells) in and around the synovial tissue. Both the FLS and the type A macrophage-like cells play an important role in the destructive aspects of the disease. The type A cells constitute the majority of the cells in normal intima and hyperplastic RA tissue. The highly invasive FLS exhibits histological features usually associated with immature tumour like fibroblasts (Qu et al., 1994; Firestein 1996). Proliferation of these synovial cells leads to pannus tissue which invades and overgrows the cartilage, leading to bone destruction (Zvaifler and Firestein, 1994). Removal of the diseased synovium is beneficial by decreasing inflammation and by preventing destruction of the proliferating pannus in adjacent structures (Thompson et al., 1973). Specific removal of this proliferating pannus tissue by a simple, non-destructive local procedure, suitable for all joints and rather specific for cells that are proliferating, is a valuable treatment for RA. (Nakamura et al., 1997; Cruz-Esteban and Wilke, 1995).
Gene therapy is a promising treatment modality for RA. Nucleic acid transfer to rheumatoid synovial tissue may result either in the production of mediators that inhibit inflammation or hyperplasia or may result in toxic substances that destroy specifically the synovium. The first clinical trials in humans were based on ex-vivo transduction of synoviocytes with IL1-RA, in order to inhibit inflammation (Evans, 1996).
The present invention was made in the course of the manipulation of adenovirus vectors. In the following section, therefore, adenoviruses are discussed.
Adenoviruses
Adenoviruses contain a linear double-stranded DNA molecule of approximately 36,000 base pairs (“bp”). It contains identical Inverted Terminal Repeats (“ITRs”) of approximately 90–140 bp, with the exact length dependent on the serotype. The viral origins of replication are within the ITRs exactly at the genome ends. The transcription units are divided in early and late regions. Shortly after infection, the E1A and E1B proteins are expressed, and function in transactivation of cellular and adenoviral genes. The early regions E2A and E2B encode proteins (DNA binding protein, pre-terminal protein and polymerase) required for the replication of the adenoviral genome (reviewed in van der Vliet, 1995). The early region E4 encodes several proteins with pleiotropic functions, for example, transactivation of the E2 early promoter, facilitating transport and accumulation of viral mRNAs in the late phase of infection and increasing nuclear stability of major late pre-mRNAs (reviewed in Leppard, 1997). The early region 3 encodes proteins that are involved in modulation of the immune response of the host (Wold et al., 1995). The late region is transcribed from one single promoter (major late promoter) and is activated at the onset of DNA replication. Complex splicing and poly-adenylation mechanisms give rise to more than 12 RNA species coding for core proteins, capsid proteins (penton, hexon, fiber and associated proteins), viral protease and proteins necessary for the assembly of the capsid and shut-down of host protein translation (Imperiale, M. J., Akusjnarvi, G. and Leppard, K. N. (1995) Post-transcriptional control of adenovirus gene expression. In: The molecular repertoire of adenoviruses I. P 139–171. W. Doerfler and P. Bohm (eds.), Springer-Verlag Berlin Heidelberg).
Interaction between Virus and Host Cell
The interaction of the virus with the host cell has mainly been investigated with the serotype C viruses Ad2 and Ad5. Binding occurs via interaction of the knob region of the protruding fiber with a cellular receptor. The receptor for Ad2 and Ad5 and probably more adenoviruses is known as the ‘Coxsackievirus and Adenovirus Receptor’ or CAR protein (Bergelson et al., 1997). Internalization is mediated through interaction of the RGD sequence present in the penton base with cellular integrins (Wickham et al., 1993). This may not be true for all serotypes, for example, serotype 40 and 41 do not contain a RGD sequence in their penton base sequence (Kidd et al., 1993).
The Fiber Protein
The initial step for successful infection is the binding of adenovirus to its target cell, a process mediated through fiber protein. The fiber protein has a trimeric structure (Stouten et al., 1992) with different lengths depending on the virus serotype (Signas et al., 1985; Kidd et al., 1993). Different serotypes have polypeptides with structurally similar N and C termini, but different middle stem regions. The first 30 amino acids at the N terminus are involved in anchoring of the fiber to the penton base (Chroboczek et al., 1995), especially the conserved FNPVYP (SEQ ID NO:39) region in the tail (Arnberg et al., 1997). The C-terminus, or “knob”, is responsible for initial interaction with the cellular adenovirus receptor. After this initial binding, secondary binding between the capsid penton base and cell-surface integrins leads to internalization of viral particles in coated pits and endocytosis (Morgan et al., 1969; Svensson and Persson, 1984; Varga et al., 1991; Greber et al., 1993; Wickham et al., 1993). Integrins are ab-heterodimers of which at least 14 a-subunits and 8 β-subunits have been identified (Hynes, 1992). The array of integrins expressed in cells is complex and will vary between cell types and cellular environment. Although the knob contains some conserved regions between serotypes, knob proteins show a high degree of variability, indicating that different adenovirus receptors exist.
Adenoviral Serotypes
At present, six different subgroups of human adenoviruses have been proposed, which in total encompass approximately 50 distinct adenovirus serotypes. Besides these human adenoviruses, many animal adenoviruses have been identified. (See, e.g., Ishibashi and Yasue, 1984).
A serotype is defined on the basis of its immunological distinctiveness as determined by quantitative neutralization with animal antisera (e.g., horse, rabbit). If neutralization shows a certain degree of cross-reactivity between two viruses, distinctiveness of serotype is assumed if A) the hemagglutinins are unrelated, as shown by lack of cross-reaction on hemagglutination-inhibition, or B) substantial biophysical/biochemical differences in DNA exist (Francki et al., 1991). The serotypes identified last (nos. 42–49) were isolated for the first time from HIV infected patients (Hierholzer et al., 1988; Schnurr et al., 1993). For reasons not well understood, most of such immuno-compromised patients shed adenoviruses that were never isolated from immuno-competent individuals (Hierholzer et al., 1988, 1992; Khoo et al., 1995).
Besides differences towards the sensitivity against neutralizing antibodies of different adenovirus serotypes, adenoviruses in subgroup C, such as Ad2 and Ad5, bind to different receptors as compared to adenoviruses from subgroup B such as Ad3 and Ad7 (Defer et al., 1990; Gall et al., 1996). Likewise, it was demonstrated that receptor specificity could be altered by exchanging the Ad3 knob protein with the Ad 5 knob protein, and vice versa (Krasnykh et al., 1996; Stevenson et al., 1995, 1997). Serotypes 2, 4, 5 and 7 all have a natural affinity towards lung epithelia and other respiratory tissues. In contrast, serotypes 40 and 41 have a natural affinity towards the gastrointestinal tract. These serotypes differ in at least capsid proteins (penton-base, hexon), proteins responsible for cell binding (fiber protein), and proteins involved in adenovirus replication. It is unknown to what extent the capsid proteins determine the differences in tropism found between the serotypes. It may very well be that post-infection mechanisms determine cell type specificity of adenoviruses. It has been shown that adenoviruses from serotypes A (Ad12 and Ad31), C (Ad2 and Ad5), D (Ad9 and Ad15), E (Ad4) and F (Ad41) all are able to bind labeled, soluble CAR (sCAR) protein when immobilized on nitro-cellulose. Furthermore, binding of adenoviruses from these serotypes to Ramos cells, that express high levels of CAR but lack integrins (Roelvink et al., 1996), could be efficiently blocked by addition of sCAR to viruses prior to infection (Roelvink et al., 1998). However, the fact that (at least some) members of these subgroups are able to bind CAR does not exclude that these viruses have different infection efficiencies in various cell types. For example, subgroup D serotypes have relatively short fiber shafts compared to subgroup A and C viruses. It has been postulated that the tropism of subgroup D viruses is to a large extend determined by the penton base binding to integrins (Roelvink et al., 1996; Roelvink et al., 1998). Another example is provided by Zabner et al., 1998 who tested 14 different serotypes on infection of human ciliated airway epithelia (“CAE”) and found that serotype 17 (subgroup D) was bound and internalized more efficiently then all other viruses, including other members of subgroup D. Similar experiments using serotypes from subgroup A-F in primary fetal rat cells showed that adenoviruses from subgroup A and B were inefficient whereas viruses from subgroup D were most efficient (Law et al., 1998). Also, in this case, viruses within one subgroup displayed different efficiencies. The importance of fiber binding for the improved infection of Ad 17 in CAE was shown by Armentano et al. (published PCT International Patent Application WO 98/22609) who made a recombinant LacZ Ad2 virus with a fiber gene from Ad 17 and showed that the chimaeric virus infected CAE more efficiently than LacZ Ad2 viruses with Ad2 fibers.
Thus, despite their shared ability to bind CAR, differences in the length of the fiber, knob sequence and other capsid proteins like penton base may determine the efficiency by which an adenovirus infects a certain target cell. Of interest is that Ad5 and Ad2 fibers bind to fibronectin III and MHC class 1 a2 derived peptides, while Ad3 fibers do not. This suggests that adenoviruses are able to use cellular receptors other than CAR (Hong et al., 1997).
Serotypes 40 and 41 (subgroup F) are known to carry two fiber proteins differing in the length of the shaft. The long shafted 41L fiber is shown to bind CAR whereas the short shafted 41S is not capable of binding CAR (Roelvink et al., 1998). The receptor for the short fiber is not known.
Adenoviral Nucleic Acid Delivery Vectors
Most adenoviral nucleic acid delivery vectors currently used in gene therapy are derived from the serotype C adenoviruses Ad2 or Ad5. The vectors have a deletion in the E1 region, where novel genetic information can be introduced. The E1 deletion renders the recombinant virus replication defective. It has been demonstrated extensively that recombinant adenovirus, in particular serotype 5, is suitable for efficient transfer of genes in vivo to the liver, the airway epithelium and solid tumors in animal models and human xenografts in immuno-deficient mice (Bout 1996, 1997; Blaese et al., 1995).
Nucleic acid transfer vectors derived from adenoviruses (“adenoviral vectors”) have a number of features that make them particularly useful for nucleic acid transfer:    1) the biology of the adenoviruses is well characterized,    2) the adenovirus is not associated with severe human pathology,    3) the adenovirus is extremely efficient in introducing its DNA into the host cell,    4) the adenovirus can infect a wide variety of cells and has a broad host-range,    5) the adenovirus can be produced at high titers in large quantities, and    6) the adenovirus can be rendered replication defective by deletion of the early-region 1 (E1) of the viral genome (Brody and Crystal, 1994).
However, there are still a number of drawbacks associated with the use of adenoviral vectors. These include:    1) Adenoviruses, especially the well investigated serotypes Ad2 and Ad5, usually elicit an immune response by the host into which they are introduced,    2) it is currently not feasible to target the virus to certain cells and tissues,    3) Some cell types are not easily transduced by the current generation of adenovirus vectors.    4) the serotypes Ad2 or Ad5 are not ideally suited for delivering additional genetic material to organs other than the liver. The liver can be particularly well transduced with vectors derived from Ad2 or Ad5. Administration of these vectors via the bloodstream leads to a significant delivery of the vectors to the cells of the liver. In therapies were cell types other than liver cells need to be transduced some means of liver exclusion must be applied to prevent uptake of the vector by these cells. Current methods rely on the physical separation of the vector from the liver cells. Most of these methods rely on localizing the vector and/or the target organ via surgery, balloon angioplasty or direct injection into an organ or a bone structure via, for instance, needles. Liver exclusion is also being practiced through delivery of the vector to compartments in the body that are essentially isolated from the bloodstream thereby preventing transport of the vector to the liver. Although these methods mostly succeed in avoiding gross delivery of the vector to the liver, most of the methods are crude and still have considerable leakage and/or have poor target tissue penetration characteristics. In some cases, inadvertent delivery of the vector to liver cells can be toxic to the patient. For instance, delivery of a HSV TK gene for the subsequent killing of dividing cancer cells through administration of GCV is not without risk when also a significant amount of liver cells are transduced by the vector. Significant delivery and subsequent expression of the HSV-TK gene to liver cells is associated with severe toxicity. Thus, there is a discrete need for an inherently safe vector provided with the property of a reduced transduction efficiency of liver cells.